Patent Application
20230372407
The human body, like that of other mammals, is colonized by trillions of microbial organisms, including bacteria, viruses, fungi, and archaea, collectively referred to as the commensal microbiota. (Erturk-Hasdemir and Kasper, 2013; Human Microbiome Project, 2012) With advances in culture-independent DNA sequencing technologies and bioinformatics, there has been a rapid increase in our knowledge of the diversity and dynamics of the microbiota over the past two decades. (Human Microbiome Project, 2012; Qin et al., 2010) Current estimates suggest that there are roughly as many bacterial cells as human cells in the body, and approximately a hundred times more bacterial genes than human genes, the vast majority of which reside in the lower gastrointestinal (GI) tract, which is colonized by an estimated 1014 bacteria. (Qin et al., 2010; Savage, 1977; Sender et al., 2016; Turnbaugh et al., 2007) Despite these advances, relatively little is understood about the molecular mechanisms by which specific commensal microbes interact with their mammalian host.
This disclosure is premised in part on the finding that Bacteroides spp. in the gut microbiome mediate a protective immune response through the TLR4 signaling pathway. This finding is surprising, in part, because it has been previously shown that the TLR4 pathway also mediates aberrant immune responses that occur in response to other bacterial species. For example, lipopolysaccharide (LPS) from E. coli also mediates its effects through engagement of TLR4 but the consequences of that engagement, such as for example sepsis, are detrimental to the host.
This disclosure further identifies that Bacteroides spp. engage TLR4 and trigger a protective immune response through the glycolipid moiety that anchors polysaccharide A (PSA) to the bacterial membrane. The downstream cascade that occurs following such TLR4 engagement by Bacteroides spp. and their components, and subsequent signaling, was also unexpected and unpredictable.
The disclosure therefore contemplates the use of the Bacteroides glycolipid in a variety of forms including an isolated form, as lipidated PSA, as an outer membrane derived from a Bacteroides sp., or as whole, inactivated Bacteroides spp., as examples.
These findings indicate that the Bacteroides glycolipid may be used in a number of clinical applications. For example, the Bacteroides glycolipid may be used in the treatment of TLR4-dependent conditions, in order to suppress or down-regulate TLR4 engagement and/or signaling. Such conditions may be currently treated (or contemplated to be treated) using a TLR4 antagonist. The Bacteroides glycolipid of this disclosure may function by competing for binding to TLR4 and redirecting the subsequent TLR4 signaling cascade towards a beneficial immune response, rather than blocking TLR4 signaling altogether. Thus, the Bacteroides glycolipid may be advantageous over previously described TLR4 antagonists in the management of TLR4-dependent conditions.
More generally, the Bacteroides glycolipid is contemplated for use in modulating TLR4 engagement and/or signaling in subjects in need thereof. Such subjects include those receiving or who have received a TLR4 agonist or antagonist. The Bacteroides glycolipid may act in these situations to modulate adverse effects of the TLR4 agonist or antagonist.
The Bacteroides glycolipid has also been shown to provide protective immunity, particularly to viral infections, in subjects who are experiencing or who are likely to experience an imbalance in the gut microbiome. Such subjects may be those who have been or will be administered a broad-spectrum antibiotic that will adversely impact the gut microbiome. Such subjects may be more susceptible to other infections such as for example viral infection. The Bacteroides glycolipid may be used in these situations in order to protect the subject from such infections during the course of treatment with the broad-spectrum antibiotic. A similar situation may present itself in subjects undergoing cancer therapy, and therefore such subjects are also contemplated for treatment with the Bacteroides glycolipid.
Thus the Bacteroides glycolipid may be used to enhance the immune response of a subject in order to provide such subject protection against a subsequent (or ongoing) viral exposure. The Bacteroides glycolipid has been shown to provide protection against two diverse viruses of different classes, and is contemplated for use as a broad spectrum anti-viral agent.
This disclosure therefore provides a method for stimulating an anti-viral immune response in a subject, comprising administering to the subject an effective amount of a Bacteroides glycolipid comprising a diglucosamine substituted with one or more acyl chains. The glycolipid may be administered on a regular basis. The immune response protects the subject against a viral infection resulting from exposure to a virus.
Also provided herein is a method for treating a subject to protect against a viral infection resulting from exposure to a virus, comprising administering to the subject an effective amount of a Bacteroides glycolipid comprising a diglucosamine substituted with one or more acyl chains, on a regular basis.
Also provided herein is a method for treating a subject, comprising administering an effective amount of a Bacteroides glycolipid comprising a diglucosamine substituted with one or more acyl chains, on a regular basis, to a subject at risk of exposure to a virus or developing a viral infection.
The glycolipid may be derived from B. fragilis or another Bacteroides spp. or it may be produced de novo in a cell free system. It may be provided in any one or a combination of a variety of forms, as provided herein.
In some embodiments, the glycolipid is administered on a regular basis. In some embodiments, a regular basis is daily, every two days, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every two weeks, or monthly.
In some embodiments, the subject is immunocompromised.
In some embodiments, the subject was recently treated with an antibiotic, optionally an antibiotic that targets anaerobic bacteria, or an antibiotic that is a broad spectrum antibiotic. In some embodiments, the subject was treated with the antibiotic in the last 2 months, in the last 1 month, or in the last week. In some embodiments, the subject is being treated with an antibiotic, optionally an antibiotic that targets anaerobic bacteria. In some embodiments, the subject will be treated with an antibiotic, optionally an antibiotic that targets anaerobic bacteria. In some embodiments, the subject will be treated with an antibiotic in the next day, in the next week, or in the next month.
In some embodiments, the subject has come into contact with a subject exposed to the virus or having the viral infection or with a bodily fluid of a subject exposed to the virus or having the viral infection. In some embodiments, the subject is present or will be present in a region in which the viral infection is endemic.
In some embodiments, the viral infection is selected from the group consisting of SARS coronavirus, influenza virus, Ebola virus, norovirus, Zika virus, West Nile virus, Dengue virus, herpesvirus, equine encephalitis virus, respiratory syncytial virus (RSV), cytomegalovirus, rabies virus, or measles virus infection.
In some embodiments, the Bacteroides glycolipid is administered orally, optionally in an enteric-coated pill, capsule or tablet.
In some embodiments, the Bacteroides glycolipid is provided in an unconjugated, cell-free and membrane-free form. In some embodiments, the Bacteroides glycolipid is provided as lipidated PSA. In some embodiments, the Bacteroides glycolipid is provided as Bacteroides outer membrane vesicle (OMV). In some embodiments, the Bacteroides glycolipid is provided as inactivated Bacteroides spp. In some embodiments, the Bacteroides glycolipid is provided as live attenuated Bacteroides spp. In some embodiments, the Bacteroides glycolipid is provided as live unattenuated Bacteroides spp.
In some embodiments, the glycolipid is not administered with a viral antigen. In some embodiments, the glycolipid is administered with a viral antigen.
Also provided is a method for modulating TLR4 signaling comprising administering to a subject in need thereof an effective amount of a Bacteroides glycolipid comprising a diglucosamine substituted with one or more acyl chains.
In some embodiments, the subject has a TLR4-mediated condition.
In some embodiments, the subject does not have sepsis.
In some embodiments, the subject is administered or exposed to a TLR4 agonist. In some embodiments, the TLR4 agonist is buprenorphine, carbamazepine, fentanyl, levorphanol, lipopolysaccharide (LPS), methadone, morphine, oxycarbezepine, oxycodone, pethidine, tapentadol, or morphine-3-glucuronide.
In some embodiments, the subject is administered or exposed to a TLR4 antagonist. In some embodiments, the TLR4 antagonist is naloxone, naltrexone, propentofylline, palmitoylethanolamide, amitriptyline, cyclobenzaprine, ketotifen, imipramine, mianserin, ibudilast, pinocembrin, or resatorvid.
These and other aspects and embodiments will be described in greater detail herein.
It is to be understood that the Figures are not necessarily to scale, emphasis instead being placed upon generally illustrating the various concepts discussed herein.
The color versions of the Figures are available in the file wrappers of the priority applications 63/039432 filed on Jun. 15, 2020, 63/039,843 filed on Jun. 16, 2020, and 63/085,392 filed on Sep. 30, 2020, the entire contents of which are incorporated by reference herein.
This disclosure is premised in part on the finding that Bacteroides spp. in the gut microbiome mediate a protective immune response through a glycolipid that engages the TLR4 signaling pathway. The glycolipid anchors polysaccharide A (PSA) to the bacterial membrane and its structure is described below. For the sake of clarity and brevity, it is referred to herein as the Bacteroides glycolipid although this does not intend that it must be produced or sourced solely from Bacteroides spp. Rather, this disclosure contemplates that the glycolipid may be generated de novo, apart from naturally occurring sources.
The Bacteroides glycolipid component minimally comprises a diglucosamine, substituted with one or more acyl chains, preferably 3, 4 or 5 acyl chains. Exemplary glycolipid structures are provided in published PCT Application No. WO2018/014012. The diglucosamine moiety may be monophosphorylated or non-phosphorylated (e.g., dephosphorylated). The various teachings provided herein, including aspects and embodiments of the invention, that are described with respect to the glycolipid are to be understood to apply equally to lipooligosaccharide (LOS, lipidated diglucosamine conjugated to an oligosaccharide, as described herein), as well as other compounds or compositions that comprise the glycolipid, including but not limited to lipidated PSA (polysaccharide A conjugated to LOS), outer membranes (OM), outer membrane vesicles (OMV), Bacteroides cells whether live or inactivated or attenuated, and the like, unless stated otherwise.
The diglucosamine may be conjugated to one or more acyl chains, including three, four, or five acyl chains in some instances via for example ester or amide linkages, and thus may be referred to as “0” substituted (or O-linked) or “N” substituted (or N-linked) respectively. In some instances, the glycolipid comprises three, four or five acyl chains. Accordingly, the glycolipid may be referred to herein as tri-acylated, tetra-acylated or penta-acylated forms.
The acyl chains may range in length from 14 to 17 carbons, in some instances. The acyl chains may be unmodified or they may be modified. If modified, the acyl chains may be hydroxy-modified. Thus, in some instances, the glycolipid may comprise one or more acyl chains characterized as C14:0, C14:0-OH, C15:0, C15:0-OH, C16:0, C16:0-OH, C17:0, and C17:0-OH. Any of the acyl chains may be branched at C13-C17.
A single preparation of glycolipid may yield a number glycolipid species that differ from each other with respect to acyl chain number. The exact composition of a glycolipid may be determined using mass spectrometry (MS), wherein different glycolipid species give rise to different and discernable spectra.
Examples of different penta-acylated species include:
Various species of tetra-acylated and tri-acylated species are similarly contemplated.
It will therefore be appreciated glycolipids of this disclosure, whether isolated from B. fragilis or generated de novo, and whether conjugated or unconjugated to an oligosaccharide core unit, may comprise any of the foregoing combinations of acyl chains, without limitation:
(1) C16:0-OH acyl chain(s) only,
The number of each type of chain may vary, and may include without limitation the following options
The foregoing examples are not to be considered limiting, and rather the disclosure contemplates various combinations, and combinations of the foregoing, to be used in compositions provided herein.
The disclosure provides compositions comprising glycolipids that are only or predominantly (e.g., greater than 50%, or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) tri-acylated, or tetra-acylated, or penta-acylated, or some combination thereof including but not limited to tetra- and penta-acylated.
The preparations provided herein can be characterized by their content of glycolipids, including those released from the tetrasaccharide repeating units of PSA or the oligosaccharide core unit of PSA. Such content can be about at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more (weight by weight of the glycolipid to the remaining components in the preparation). The amount of glycolipid may be determined for example using the gel electrophoresis methods or mass spec methods.
The purity of the glycolipid may also be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, or higher (weight by weight of the glycolipid to other Bacteroides-derived components in the preparation, wherein such components are unconjugated to the glycolipid).
Isolated intends that the glycolipid (or other agent) is provided in a more pure form or a more concentrated form compared to its synthesized or naturally occurring form. An “isolated” glycolipid may be a glycolipid that is prepared or obtained from a Bacteroides cell, and is physically separated from a natural environment (e.g., a Bacteroides cell, components of the Bacteroides cell, and/or components of the Bacteroides cell capsular complex such as but not limited to PSB).
In some embodiments, the compositions are substantially free of naturally occurring contaminants such as nucleic acids (e.g., DNA and RNA), proteins, and other components of a Bacteroides cell and/or a Bacteroides capsule. Substantially free, as used herein, intends that these contaminants represent about or less than 5%, less than 1%, less than 0.5%, or less than 0.1% (or less) by weight (weight of the contaminant to weight of the glycolipid). In some instances, such contaminants may be undetectable.
The glycolipid may be conjugated to an oligosaccharide, referred to herein as an oligosaccharide core unit, at one of the glucosamine residues. Such oligosaccharide may comprise 10 or fewer sugars. The sugars may be galactose, glucose and/or fucose. The oligosaccharides are typically conjugated to the diglucosamine via an acid-labile linkage such as but not limited to a ketosidic linkage.
The Bacteroides glycolipid may be provided in a number of forms. Those forms include the glycolipid form (i.e., the acyl-substituted diglucosamine) free of the oligosaccharide core unit or the tetrasaccharide unit(s) of PSA, the glycolipid conjugated to the oligosaccharide core unit, the glycolipid conjugated to the oligosaccharide core unit and to the tetrasaccharide unit(s) (i.e., lipidated PSA with varying polysaccharide lengths). The oligosaccharide core unit, by virtue of its hydrophilicity, helps to render the glycolipid more water soluble, therefore in some instances the glycolipid is conjugated to the oligosaccharide core unit. In addition, any of the foregoing forms may be provided in a cell-free form or a membrane-free form, or alternatively they may be provided bound to a membrane, such as an outer membrane, or bound to a cell such as a Bacteroides cell such as B. fragilis. If provided as a Bacteroides cell such as B. fragilis, such cells may be inactivated or attenuated. Alternatively, it may be live and unattenuated (such as in a live active culture), and in this form in some instances may be provided in a foodstuff such as a probiotic (e.g., in a yogurt, supplement, etc.).
The glycolipid may be obtained from Bacteroides spp., including but not limited to B. fragilis. One source, for example, may be a mutant non-naturally occurring strain of B. fragilis that overexpresses PSA relative to other polysaccharides. Another source may be a mutant non-naturally occurring strain of B. fragilis that expresses only PSA.
The Bacteroides glycolipid may be extracted from Bacteroides spp. using a phenol/water extraction, and DNase, RNase and pronase treatments. The subsequent extraction steps influence whether the glycolipid is harvested in a conjugated or an unconjugated form. For example, if the material is then treated with deoxycholate (DOC), the polysaccharide moiety (i.e., the repeating tetrasaccharide moiety) of PSA is removed, and the resultant material is the glycolipid conjugated to the oligosaccharide core unit. The oligosaccharide core unit, by virtue of its hydrophilicity, helps to render the lipid component more water soluble. If the material is treated instead or in addition with acid hydrolysis, the acid-labile linkage between the oligosaccharide core unit and the acyl-substituted diglucosamine is cleaved and the acyl-substituted diglucosamine alone is obtained.
Polysaccharide A
The polysaccharide component of lipidated PSA, referred to herein as PSA, comprises a tetrasaccharide repeating unit shown below. It possesses zwitterionic behavior as conferred by a positive charge on its free amine group and a negative charge on its free carboxyl group (per repeating tetrasaccharide unit). Its naturally occurring state has been reported to comprise over 60 tetrasaccharide repeating units (e.g., up to and including in some instances about 100, or about 200, or about 300 repeated units on average), and it has an average molecular size of about 150 kD (with a range of about 75 kD to 240 kD).
The repeating tetrasaccharide unit of PSA has a structure as follows:
The tetrasaccharide repeating unit may also be expressed as follows:
Synthetic forms of lipidated PSA may comprise comprising various ranges of tetrasaccharide units (e.g., 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, or 1-5 tetrasaccharide units). Such shorter variants can be obtained by depolymerizing naturally occurring lipidated PSA or by depolymerizing PSA obtained from lipidated PSA. PSA can be depolymerized using for example chemical means (e.g., using reactive oxygen species or reactive nitrogen species such as but not limited to nitrogen monoxide, as described in Duan and Kasper, Glycobiology, 2011, 21(4):401-409), mechanical means, and/or enzymatic means that are known in the art.
Also contemplated are synthetic forms of lipidated PSA comprising more than 300 repeating tetrasaccharide units, including without limitation 350, 400, 500, 600, 700, 800, 900 or 1000 units or more.
The polysaccharide component may be covalently conjugated to the glycolipid, or in certain synthetic forms it may be unconjugated to the glycolipid. If covalently conjugated, it may be conjugated via a glycosidic bond to the oligosaccharide core unit. In other embodiments, it may be conjugated via a ketosidic bond or other acid labile bond or via a bond such as an ester, an amide, or an ether bond to form a non-naturally occurring lipidated PSA.
The glycolipids may also be provided in the form of outer membrane vesicles (OMV). In this form, the glycolipids may be provided as lipidated PSA that is anchored in the OMV or they may be provided anchored in the OMV in another manner. Methods for making OMV from bacterial species, including Bacteroides spp., are provided in US Published Applications 2013/0121966, 2015/0017664, and 2017/0145061.
Further, the glycolipids may be provided in the form of intact bacterial cells, and such cells may be live, active cells (as in a live active bacterial culture), or they may be inactivated and/or attenuated.
The glycolipids and compositions thereof may be administered to a subject having or at risk of having a viral infection. The glycolipids and compositions thereof may be used and/or administered with another therapeutic such as an anti-viral agent. Anti-viral agents include immunoglobulin, amantadine, nucleoside analogue, nonnucleoside analogue, biflavanoid and protease inhibitor. In one embodiment, the protease inhibitor is indinavir, saquinavir, ritonavir, and nelfinavir. In another embodiment, the biflavanoid is robustaflavone, amentoflavone, or a derivative or salt thereof. In yet another embodiment, the non-nucleoside analogue is selected from the group consisting of delavirdine, nevirapine, efavirenz, alpha-interferon, recombinant CD4, amantadine, rimantadine, ribavirin and vidarabine. The anti-viral may be remdesivir.
The virus may be a picornavirus (e.g., polio virus, foot and mouth disease virus), calicivirus (e.g., norovirus), togavirus (e.g., sindbis virus, the equine encephalitis viruses, chikungunya virus, rubella virus, Ross River virus, bovine diarrhea virus, hog cholera virus), flavivirus (e.g., dengue virus, West Nile virus, yellow fever virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus), coronavirus (e.g., SARS, MERS, SARS-CoV-2, SARS-CoV-1, human coronaviruses (common cold), swine gastroenteritis virus), rhabdovirus (e.g., rabies virus, vesicular stomatitis viruses), filovirus (e.g., Marburg virus, Ebola virus), paramyxovirus (e.g., measles virus, canine distemper virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, Newcastle disease virus, rinderpest virus), orthomyxovirus (e.g., human influenza viruses, avian influenza viruses, equine influenza viruses), bunyavirus (e.g., hantavirus, LaCrosse virus, Rift Valley fever virus), arenavirus (e.g., Lassa virus, Junin virus (JUNV), Machupo virus), reovirus (e.g., human reoviruses, human rotavirus), birnavirus (e.g., infectious bursal virus, fish pancreatic necrosis virus), retrovirus (e.g., HIV-1, HIV-2, HTLV-1, HTLV-2, bovine leukemia virus, feline immunodeficiency virus, feline sarcoma virus, mouse mammary tumor virus), hepadnavirus (e.g., hepatitis B virus), parvovirus (e.g., human parvovirus B, canine parvovirus, feline panleukopenia virus), papovavirus (e.g., human papillomaviruses (HPV), SV40, bovine papillomaviruses), adenovirus (e.g., human adenovirus, canine adenovirus, bovine adenovirus, porcine adenovirus), herpes virus (e.g., herpes simplex viruses such as HSV-1 and HSV-2, varicella-zoster virus, infectious bovine rhinotracheitis virus, human cytomegalovirus, human herpesvirus 6), poxvirus (e.g., vaccinia, fowlpoxviruses, raccoon poxvirus, skunkpox virus, monkeypoxvirus, cowpox virus, musculum contagiosum virus), poliovirus or rhinovirus.
The virus may be severe acute respiratory syndrome (SARS) causing virus, Middle East respiratory syndrome (MERS) causing virus, severe acute respiratory syndrome coronavirus 2 (also known as SARS-CoV-2 and 2019-nCoV, cause of COVID-19), or, SARS-CoV-1.
The virus may be hepatitis A virus (HAV), hepatitis C virus (HCV), Epstein Barr Virus (EBV), or Rous Sarcoma Virus (RSV), Enterovirus 71 (EV71), Coxsackieviruses, including serotypes A1, A4, A6, A10, and A16, Human enterovirus A (HEA), pestiviruses, measles, smallpox, cowpox, Vesicular Stomatitis Virus (VSV), Zika virus, HIV, Herpes simplex virus 1, Herpes simplex virus 2, cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human papilloma virus, rotavirus, adenovirus, influenza A virus, respiratory syncytial virus, varicella-zoster virus, small pox, monkey pox.
In some embodiments, the virus is a coronavirus, flavivirus, hepadnavirus, herpesvirus, orothomyxovirus, filovirus, arenavirus, calcivirus, togavirus or paramyxoviruses.
In some embodiments, the virus is SARS coronavirus, influenza virus, Ebola virus, norovirus, Zika virus, West Nile virus, Dengue virus, herpesvirus, equine encephalitis virus, respiratory syncytial virus (RSV), cytomegalovirus, rabies virus, or measles virus.
The subject may be administered the glycolipid repeatedly during a period of time or situation in which the subject is likely to be exposed to a virus. For example, the glycolipid may be administered daily, every 2, 3, 4, 5, 6 day, weekly, every 2, 3 weeks or monthly. If the subject has been exposed to a virus, or a subject who has been exposed to the virus, then the subject may be treated immediately and regularly after such exposure for a period of time. The period of time may be the incubation period of the virus (i.e., the average time it takes for symptoms to appear after exposure). The subject may be treated ahead of contact with another who is carrying the virus or has likely been exposed to the virus, and such treatment may continue beyond such contact, including for the length of the incubation period to ensure the subject manifests no symptoms associated with an infection by the virus (e.g., a fever and/or chills, muscle aches, coughing, respiratory difficulty, aberrant cardiovascular manifestations such as aberrant clotting, headaches, rashes, etc.).
The subject to be treated may therefore manifest no symptoms ahead of treatment. The subject may be tested for the presence of the virus or antibodies to the virus following treatment and/or following the incubation period. A subject who has been treated may be negative for the virus even though the subject has been in contact with the virus. A subject who has been treated may be positive for the virus but may have experienced no or diminished symptoms associated with the virus, particularly during the incubation period. A subject who has been treated may be positive for anti-viral antibodies but may have experienced no or diminished symptoms associated with the virus, particularly during the incubation period. Diminished symptoms include symptoms of lesser severity and/or of shorter duration than those experienced by untreated and infected subjects.
In some instances, the glycolipid is administered in amounts effective to induce IFNbeta in the subject at a level and for a time considered to be protective. Thus, the subject may be administered the glycolipid throughout a time during which the subject is more likely to be exposed to the virus, and the glycolipid is administered in an amount to induce sufficiently protective levels of IFNbeta. IFNbeta levels may be measured from blood samples of the subject.
In some embodiments, the subject to be treated is at a risk of exposure to the virus that is higher than the risk of exposure of the general public. Such subjects may include those medical professionals, hospital workers, and those who are hospitalized.
In some embodiments, the subject to be treated is at risk of exposure to one more than one virus, and optionally the risk is higher than the risk of exposure to the general public. Such subjects may include those medical professionals, hospital workers, and those who are hospitalized.
In some embodiments, the subject to be treated is at risk of developing an infection upon exposure to a virus, and optionally the risk is higher than the risk of developing an infection by the general public. Such subjects may include those who were, are and/or will be treated with one or more antibiotics and/or with a broad spectrum antibiotic. Such subjects may include those who have received another therapy that has caused imbalance in their gut microbiome, such as but not limited to a cancer chemotherapy.
This disclosure further provides methods for treating TLR4-dependent conditions. TLR4-dependent conditions are conditions that are mediated at least in part by TLR4 signaling. Such signaling may be aberrant or uncontrolled or may lead to downstream events that are detrimental to the subject. Examples of TLR4-dependent conditions include bacterial infections, sepsis, organ fibrosis, certain inflammatory conditions, and others known in the art. In some instances, the TLR4-dependent condition is not sepsis. In some instances, the subject does not have sepsis. In some instances, the TLR4-dependent condition is not an autoimmune disorder. In some instances, the subject does not have an autoimmune disorder.
The glycolipids of this disclosure may be used in such subjects to modulate TLR4 signaling in quality and/or quantity. The glycolipids may be used to engage TLR4, thereby competing with the condition-causing agent for binding and signal induction through TLR4, with the glycolipids inducing a different downstream response than the one induced by the condition-causing agent. In such situations, the glycolipids may be used to reduce or eliminate the side effects of the condition-causing agent. The glycolipids may be administered once or repeatedly while the condition is manifested. The administration regimen of the glycolipids may also depend on the nature of the condition and the condition-causing agent. If the condition-causing agent is a TLR4 agonist or antagonist that is deliberately administered to the subject, then the glycolipid may be administered alongside of or at a spaced time interval from the TLR4 agonist or antagonist.
In related aspects, the glycolipids may be used to modulate TLR4 signaling. The glycolipids may be used to reduce signaling through TLR4 in subjects who are experiencing elevated TLR4 signaling, such as may be the case if the subject is being administered a TLR4 agonist. The glycolipids may be used to increase signaling through TLR4 in subjects who are experiencing reduced TLR4 signaling, such as may be the case if the subject is being administered a TLR4 agonist.
The TLR4 agonist or antagonist may be inducing signal through the MyD88-dependent pathway following TLR4 engagement. The glycolipids of this disclosure may cause a shift towards the TRIF pathway, thereby reducing signaling through MyD88 and its aberrant effects.
The subject may be one who has been administered, or is being administered, or will be administered a TLR4 agonist or antagonist. TLR4 agonists and TLR4 antagonists may be compounds that are prescribed and intended to treat another condition but which adversely impact TLR4 signaling through TLR4 engagement. Examples of TLR4 agonists are buprenorphine, carbamazepine, fentanyl, levorphanol, lipopolysaccharide (LPS), methadone, morphine, oxycarbezepine, oxycodone, pethidine, tapentadol and morphine-3-glucuronide. Examples of TLR4 antagonists are naloxone, naltrexone, propentofylline, palmitoylethanolamide, amitriptyline, cyclobenzaprine, ketotifen, imipramine, mianserin, ibudilast, pinocembrin and resatorvid.
In still other instances, the glycolipid may be used to stimulate or enhance a TLR4-mediated immune response. This may benefit subjects who fail to induce such immune responses in the absence of the glycolipid, or who need a boost in order to do so. The glycolipid may be used, in any one or combination of its forms, as a vaccine adjuvant. Additionally, the glycolipid may be used to induce immunosuppressive or immunoregulatory activity via TLR4-mediated signaling. Further data (not shown) evidence the ability of the glycolipid to induce a variety of these downstream effects, including for example regulating IL-10 secretion by T cells through TLR4/IFNb through induction of a tolerogenic DC phenotype and type I regulatory T (Tr1) cells. Tr1 cells regulate tolerance towards antigens of any origin. Tr1 cells are self or non-self antigen specific and they play a role in inducing and maintaining peripheral tolerance and suppressing tissue inflammation in autoimmunity and graft vs. host disease. It was found that PSA signals through IFNβ to induce an immunoregulatory phenotype in antigen presenting cells (APCs) and enhance IL-10 secretion by CD4+ T cells. Importantly, the protective effects of PSA in a mouse autoimmune disease model were dependent on IFN-I signaling, demonstrating the important tolerogenic role of commensal-induced IFNβ. These activities indicate that the glycolipids can be used for a variety of applications, prophylactically or therapeutically.
The methods provided herein are to use in a subject. As used herein, a “subject” may be a human or a non-human subject. Non-human subjects include, for example, agricultural animals such as cows, pigs, horses, goats, sheep, bison, etc., and domesticated animals such as dogs and cats, or other domesticated animals. In certain embodiments, the subject is human.
In some embodiments, the subject is one who has been administered, and/or is being administered, and/or will be administered a broad-spectrum antibiotic or other treatment that will create an imbalance in the gut microbiome. A broad spectrum antibiotic as used herein in an antibiotic that acts against more than one bacterial species. Some broad spectrum antibiotics act on a variety of bacterial species including both gram-positive and gram-negative species. Examples include aminoglycosides (except for streptomycin), ampicillin, amoxicillin/clavulanic acid (Augmentin), carbapenems (e.g. imipenem), piperacillin/tazobactam. quinolones (e.g. ciprofloxacin), tetracyclines, chloramphenicol, ticarcillin, and trimethoprim/sulfamethoxazole (Bactrim).
The terms “treat,” “treating” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound of this disclosure to the subject.
The glycolipids of this disclosure may be used to reduce onset of a viral infection and optionally a condition caused by the viral infection. The glycolipids and compositions thereof may be capable of reducing or preventing onset of a viral infection or side effect of exposure caused by one or more viruses when administered to a subject.
The glycolipids of this disclosure may be formulated as pharmaceutical compositions that include a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable carrier” as used herein is intended to include diluents such as saline and aqueous buffer solutions.
The glycolipids and compositions (e.g. pharmaceutical compositions) thereof can be administered to a subject by any known manner, for example, subcutaneous, intramuscular, intravenous, intradermal, by oral administration, inhalation, transdermal application, intravaginal application, topical application, intranasal or rectal administration. In one embodiment, the compound may be administered intranasally, such as inhalation.
In certain embodiments, compositions disclosed herein are administered orally or into the gastrointestinal tract for example using enteric-coated pills, capsules or tablets. The compositions, including the glycolipids in conjugated or unconjugated form, in lipidated PSA form, in the form of Bacteroides outer membrane, or in inactivated or attenuated Bacteroides cells, may be formulated as food, including yogurts, nutritional bars, and the like.
The glycolipids and/or compositions thereof may be provided in a kit for in vitro and/or in vivo use. The kits may include a suitable container, the glycolipid composition, and one or more additional agents such as other anti-viral agents. The compositions may be partially or wholly dehydrated and/or in a ready to use form. Kits may be stored at room temperatures or at refrigerated temperatures depending on the particular formulation.
The following Examples are included for purposes of illustration and are not intended to limit the scope of the invention.
The type I interferon (IFN-I) response is a crucial mediator of antiviral immunity and homeostatic immune system regulation. However, the source of IFN-I signaling under homeostatic conditions remains unclear. In this study, we discovered that the commensal microbiota regulates the IFN-I response both locally and systemically through induction of IFNβ by colon lamina propria dendritic cells. Moreover, the molecular mechanism by which a specific commensal microbe induces IFNβ was identified. Indeed, we found that outer membrane-associated glycolipids of human gut commensal microbes belonging to the Bacteroidetes phylum induce expression of IFNβ. Using Bacteroides fragilis, as well as its outer membrane associated polysaccharide A as the paradigm, we determined that IFNβ expression was induced via the TLR4-TRIF signaling pathway. Antiviral activity of this purified microbial molecule against infection with either vesicular stomatitis virus (VSV) or influenza was demonstrated to be dependent on the induction of IFNβ. In an in vivo model of VSV infection, we discovered that commensal-induced IFNβ modulates the health of the host by regulating natural resistance to virus infection. Due to the importance of IFN-Is in antiviral immunity and other vital aspects of human physiology, discovery of an IFNβ-inducing microbial molecule represents a potential novel therapeutic approach for the treatment of a variety of human diseases.
One mechanism by which microbes can modulate the host immune system is through regulation of cytokine signaling. Type I interferons (IFN-Is) are a family of structurally similar cytokines, consisting primarily of two different classes of proteins, interferon-α (IFNα) and interferon-β(IFN-β), all of which signal through the common type I interferon receptor (IFNAR). (Theofilopoulos et al., 2005) IFN-I expression is regulated at the transcriptional level, and can be induced at high levels in all nucleated cells in response to sensing of pathogens by pattern recognition receptors (PRRs). (Ivashkiv and Donlin, 2014) Indeed, IFN-Is play a critical role in the response to the majority of virus infections, through induction of a restrictive anti-viral state in cells, induction of apoptosis in virus-infected cells, and through regulation of immune cell subsets crucial to the antiviral response. (McNab et al., 2015; Muller et al., 1994; Stetson and Medzhitov, 2006; Yan and Chen, 2012) Several recent studies have demonstrated that IFN-Is are not just present during infection, but are expressed constitutively at low-levels and possess vital homeostatic functions. (Gough et al., 2012; Kole et al., 2013) Indeed, IFN-Is have the capacity to regulate the development and/or activation of virtually every immune effector cell, playing important roles in the anti-inflammatory and anti-tumor responses. (Gough et al., 2012) Despite this importance, the mechanism by which IFN-Is are induced and regulated in the absence of infection has yet to be elucidated. Indeed, research on microbial regulation of the IFN-I response has been largely limited to diseased states or administration of exogenous, and often synthetic, microbial ligands.
Due to the importance of IFN-Is to the health of the organism, both in defense against pathogens and maintenance of homeostasis, it is of critical importance to gain a mechanistic understanding of how the constitutive IFN-I response is regulated in healthy individuals and might be disrupted leading to disease, with the potential to lead to new therapeutic strategies. We hypothesized that the commensal microbiota supplies PRR signaling to regulate the IFN-I response in the absence of infection. In this study, we discovered that the commensal microbiota regulates the IFN-I response both locally and systemically through induction of IFNβ by colonic lamina propria (LP) dendritic cells (DCs), enhancing resistance to virus infection. Moreover, the molecular mechanism by which a specific commensal microbe induces IFNβ was identified. Indeed, we found that outer membrane-associated glycolipids of human gut commensal microbes belonging to the Bacteroidetes phylum induce expression of IFNβ. Using Bacteroides fragilis (B. fragilis) as the paradigm, we determined that signaling to initiate IFNβ expression was through the toll-like receptor 4-(TLR4) TIR-domain-containing adapter-inducing interferon-β (TRIF) pathway.
To investigate the role of the commensal microbiota in the host IFN-I response, the effect of microbiota depletion on downstream interferon stimulated gene (ISG) expression in various murine tissues was analyzed. The vast majority of commensal microbes reside in the GI tract. (Savage, 1977; Turnbaugh et al., 2007) Analysis was therefore performed to determine effects of the microbiota on ISG expression in the intestinal immune compartment, in the mesenteric lymph nodes (mLN), and systemically, in the spleen. Age- and gender-matched wild type (WT) specific pathogen free (SPF) C57BL/6 mice were administered either vehicle control or a cocktail of broad-spectrum antibiotics (ABX) in the drinking water for 7 days to deplete the microbiota. The relative expression levels of a panel of ISGs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A reduction in ISG expression was observed in both the mLN and spleen of WT ABX mice compared to WT SPF mice (
Antibiotics can have off-target effects, acting in some instances directly on the host rather than on the commensal microbes themselves. (Morgun et al., 2015) A second method of microbiota depletion was therefore used to confirm a direct role of the commensal microbiota on the observed phenotype. Splenic and mLN ISG expression was analyzed in WT C57BL/6 germ free (GF) mice devoid of all microbes compared to WT C57BL/6 SPF mice. Consistent with the antibiotics-treated mice, a reduction in mLN and splenic ISG expression was observed (
Of the mammalian IFN-Is, IFNβ is unique with respect to its homeostatic functions and expression pattern. (Schreiber and Piehler, 2015) It was therefore hypothesized that the commensal microbiota induces the constitutive IFN-I response, as evidenced by ISG expression, specifically through regulation of IFNβ. ISG expression levels were compared by qRT-PCR between WT and IFNβ deficient (Ifnb1−/−) SPF C57BL/6 mice. A significant reduction in ISG expression was observed in the mLN and spleen of Ifnb1−/− mice compared to WT mice (
Further data (not shown) support the finding that glycolipids of this disclosure modulate a cluster of genes that are functionally relevant to anti-viral immunity. Among various genes examined, it was found that activation of splenic dendritic cells (DC) by tri-acylated and tetra-acylated monophosphorylated glycolipids provided herein resulted in the induction of a cluster of genes that are found in ISG modules, including for example Oas1a/b, Oasl1/2, Ifit1/2, Mx1/2, and Stat1/2. These gene sets are all associated with transcriptomic changes in response to type I IFN in DCs and plasmacytoid DCs. These data strongly suggest that immunomodulatory changes driven by under-acylated monophosphorylated glycolipids could lead to anti-viral immune responses mediated by type I IFNs (e.g., IFN alpha and/or IFN beta) via IFNAR1 and cellular interaction of DCs with T cells.
To investigate the source of commensal-induced IFNβ under homeostatic conditions, flow cytometric analysis was performed on tissues harvested from IFNβ-yellow fluorescent protein (YFP) reporter mice, in which cells activated to express IFNβ express YFP. (Scheu et al., 2008) By analyzing IFNβ-YFP expression in the colon LP, mLN, and spleen, IFNβ expression was found to be localized to the intestinal milieu, with significantly higher percentages of IFNβ-YFP+CD45+ leukocytes in the colonic LP compared to the spleen or mLN (
As in other tissues, conventional DCs (cDCs) in the colon LP represent a heterogeneous population that can be classified into two subsets based on surface marker expression. cDC1 cells are CD103+CD11b− while cDC2 cells comprise both CD103−CD11b+ and CD103+CD11b+ cells. (Stagg, 2018) To determine which DCs express IFNβ, expression of CD103 and CD11b by the IFNβ-YFP+CD11c+CD45+ colonic dendritic cells was analyzed using flow cytometry. IFNβ expression was specific to the cDC2 subset of cells, including both CD103+CD11b+ and CD103-CD11b+ cells (
To confirm these findings, a second experimental approach was used in which DCs were isolated from the colon LP, mLN, and spleens of WT SPF mice using a magnetic bead-based cell separation method. Expression of Ifnb1 was analyzed in the whole tissue single cell suspension as well as both the DC+ and DC− fractions of these tissues by qRT-PCR. There was no detectable Ifnb1 expression in the whole tissue or the DC− fraction of the majority of colon LP samples (
To test the role of the microbiota in colonic DC IFNβ expression, two methods of microbiota depletion were employed. Broad-spectrum antibiotics were administered in the drinking water of IFNβ-YFP mice for 7 days, followed by flow cytometric analysis of colon LP cells. A significant reduction in the frequency of IFNβ-YFP+CD11c+ cells was observed in IFNβ-YFP mice after ABX treatment (
Having identified effects of commensal-induced IFNβ on ISG expression not just locally, in the mLN, but also systemically in the spleen, we investigated IFNβ levels in systemic circulation, by measuring serum IFNβ levels using an enzyme-linked immunosorbent assay (ELISA). Consistent with reports that IFN-I expression is extremely low under homeostatic conditions, we were unable to detect IFNβ in the serum of WT SPF or ABX mice (
Bacteroides fragilis Induces Expression of IFNβ by Colon LP DCs
Of the trillions of commensal bacteria that inhabit the mammalian gut, one of the most prominent phyla represented is the Bacteroidetes, consisting of obligate anaerobic, Gram-negative rods, with about 25% of all colonic anaerobes belonging to the genus Bacteroides within this phylum. (Salyers, 1984; Wexler, 2007) Recently an association was reported between several species of the genus Bacteroides, pDC numbers, and a IFN-I response gene signature. (Geva-Zatorsky et al., 2017) One of the Bacteroides sp. investigated in this study was B. fragilis, a common human colonic commensal bacterial species with previously demonstrated immunomodulatory properties. (An et al., 2014; Dasgupta et al., 2014; Mazmanian et al., 2005; Mazmanian et al., 2008) We hypothesized that B. fragilis regulates colon DC IFNβ expression and thus regulates the IFN-I response. GF mice were colonized with B. fragilis strain NCTC 9343 at 4 weeks of age, followed by isolation of colon LP DCs after 2 weeks and analysis of Ifnb1 expression. As a colonization control, GF mice were colonized with a different bacterial species with demonstrated immunomodulatory properties, Clostridium ramosum (C. ramosum), thus controlling for any non-specific effects due to bacterial colonization. (Geva-Zatorsky et al., 2017; Sefik et al., 2015) We observed that colonization with B. fragilis significantly enhanced Ifnb1 expression in colon LP DCs, while C. ramosum colonization had no effect (
B. fragilis Capsular Polysaccharide a Induces IFNβ Expression
The outer membrane (OM) of Gram-negative bacteria, such as B. fragilis, serves as the site of interaction between the bacterial cell and its environment and comprises several classes of potential immunomodulatory molecules. (Kasper and Seiler, 1975; May and Grabowicz, 2018) To test whether an OM component is responsible for IFNβ expression, an in vitro model was developed in which bone marrow-derived dendritic cells (BMDCs) isolated from WT mice were treated with OM complexes isolated from B. fragilis, followed by ELISA analysis of secreted IFNβ in the supernatants. It was found that B. fragilis OM complexes significantly induced IFNβ secretion by BMDCs (
To confirm the ability of PSA to induce a productive IFN-I response through IFNβ, expression of downstream ISGs was analyzed by qRT-PCR in BMDCS treated for 24 hrs with PSA or vehicle control. An increase in expression of a panel of ISGs was observed in WT BMDCs treated with PSA (
To confirm the relevance of these findings in vivo, Ifnb1 expression by colon LP DCs was analyzed in WT SPF mice administered 150 ug of PSA by oral gavage. A significant increase in Ifnb1 expression was observed 1.5 hrs after treatment (
B. fragilis Lipooligosaccharide Signals Through TLR4 to Induce IFNβ
PSA has been previously demonstrated to signal via TLR2/1 heterodimers and dectin-1 on dendritic cells to induce secretion of IL-10 by CD4+ T cells. (Dasgupta et al., 2014; Erturk-Hasdemir et al., 2019; Round and Mazmanian, 2010) The role of TLR2 and dectin-1 in PSA induction of IFNβ was therefore investigated by comparing the response of WT, dectin-1 (Clec7c−/−), and TLR2 (Tlr2−/−) deficient BMDCs to PSA using ELISA to detect IFNβ secreted in the supernatants. Much to our surprise, loss of dectin-1 or TLR2 had no effect on IFNβ secretion (
The majority of IFN-I inducing PRRs sense viral nucleic acid, with the bacteria-sensing TLR2, dectin-1, and TLR4 pathways representing some of the few exceptions. TLR4 senses microbial glycolipids and of the IFN-Is predominantly induces IFNβ, consistent with the signaling observed by PSA. (Lu et al., 2008; Sheikh et al., 2014) TLR4 deficient (Tlr4−/−) BMDCs completely lost the ability to secrete IFNβ in response to PSA treatment (
TLR4 signaling proceeds through two different signaling adapter molecules, myeloid differentiation primary response protein (MyD88) and TRIF, which activate divergent pathways to mediate downstream gene expression changes. MyD88 canonically activates the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) to induce proinflammatory gene expression while TRIF is reported to activate interferon regulatory factor 3 (IRF3) to mediate IFNβ expression. (Maeshima and Fernandez, 2013) Consistent with these well-described signaling events, PSA induction of IFNβ was entirely dependent on TRIF, as evidenced by the lack of increased IFNβ secretion in TRIF deficient (Trif−/−) BMDCs in response to PSA (
The canonical TLR4 ligand is LPS, the primary component of the OMs of Gram-negative bacteria. (Maeshima and Fernandez, 2013) In contrast to the archetypal Gram-negative bacterial species Escherichia coli (E. coli), the OM of B. fragilis lacks LPS but instead consists of a lipooligosaccharide (LOS), comprising a lipid A domain linked to a short oligosaccharide. (Lindberg et al., 1990) We recently identified that PSA contains a glycolipid portion, which structurally resembles B. fragilis LOS, and now believe that the polysaccharide domain of PSA is covalently linked to LOS as a means of attachment to the OM. (Erturk-Hasdemir et al., 2019) The lipid A component of the LOS of Bacteroides has structural and biological features which distinguish it from the much studied lipid A of E. coli. (Erturk-Hasdemir et al., 2019; Kasper, 1976) It was therefore hypothesized that the LOS portion of PSA is responsible for induction of IFNβ. Consistent with this hypothesis, purified B. fragilis LOS was observed to induce IFNβ in a dose-dependent manner through TLR4 (
LOS-like molecules are not unique to B. fragilis, and multiple bacterial species of the genus Bacteroides have been demonstrated to have LPS or LOS molecules consisting of glycolipids that are structurally similar to B. fragilis LOS and distinct from classical E. coli LPS. (Cullen et al., 2015; Kasper, 1976; Vatanen et al., 2016) We therefore hypothesized that induction of IFNβ by Bacteroides LOS/LPS represents a broader mechanism by which commensal microbes are capable of influencing the host immune system. OM complexes were isolated from several different species of Bacteroides to test their effect on cytokine secretion by BMDCs. It was observed that OMs extracted from all of the Bacteroides sp. tested induced IFNβ by BMDCs in a TLR4-dependent manner (
To address the role of Bacteroides sp. and TLR4 signaling in regulation of the IFN-I response in vivo, ISG expression was compared in WT SPF mice, Tlr4−/− SPF mice, and WT SPF mice 7 days post treatment with metronidazole, a bactericidal antibiotic which specifically targets anaerobic bacteria, including Bacteroides sp. (Edwards, 1979; Salyers, 1984) WT mice treated with metronidazole alone and Tlr4−/− SPF mice both exhibited significant decreases in ISG expression in the mLN (
It was hypothesized that commensal-induced IFNβ is functionally important for the priming of the antiviral response and resistance to virus infection. A murine vesicular stomatitis virus (VSV) infection model, which causes meningoencephalitis and fatal paralytic disease, was used to investigate the role of commensal-induced IFNβ. To test the contribution of IFNβ to protection in this disease model, cohoused Ifnb1+/+ and Ifnb1−/− littermates were infected by subcutaneous injection into the footpad with 106 plaque-forming units (PFU) of VSV strain Indiana. Ifnb1−/− mice exhibited increased susceptibility to disease based on all parameters tracked, with increased daily and cumulative disease scores (
It is important to note that VSV infection itself induces IFNβ expression through PRR signaling, which likely contributes to ISG expression after infection and antiviral immunity. (Kato et al., 2005) Therefore, the increased susceptibility observed in the Ifnb1−/− mice likely reflects virus infection-induced IFNβ in addition to any contributions of priming levels of IFNβ induced by commensal microbes prior to infection. To investigate the effects of priming of the antiviral response by constitutive, microbiota-induced IFNβ, an IFNβ neutralizing antibody (anti-IFNβ) was used to selectively inhibit IFNβ prior to infection. (Sheehan et al., 2015). WT mice were administered two doses of anti-IFNβ or mouse IgG2a isotype control antibody by IP injection, 72 hrs and 24 hrs prior to infection with 106 PFU of VSV. Anti-IFNβ treated mice had increased severity of disease (
To further investigate the impact of the commensal microbiota on VSV infection, WT mice were administered broad-spectrum antibiotics for 7 days prior to infection, stopping on the day of infection. Increased susceptibility to disease was observed in WT ABX mice compared to WT SPF mice, with significantly increased paralysis scores (
B. fragilis PSA Possesses IFNβ-Mediated Antiviral Activity
To further investigate the antiviral activity of commensal-induced IFNβ, we sought to identify whether signaling of a purified IFNβ-inducing commensal microbial molecule, B. fragilis PSA, is sufficient to protect against virus infection. While LOS and the LOS domain of PSA are responsible for IFNβ induction, whole PSA was used in this system due to its enhanced solubility. An in vitro virus infection model was developed in which BMDCs were treated with a dose response of PSA for 24 hrs prior to infection with a green fluorescent protein (GFP) expressing strain of VSV Indiana (VSV-GFP) at a multiplicity of infection (MOI) of 1, followed by flow cytometric analysis of the percentage of infected (GFP+) live cells 24 hrs post infection (h.p.i.) (
To identify whether the observed in vitro protective effects of PSA are limited to VSV infection, a second virus infection model was developed, using the murine adapted influenza A virus (IAV) strain PR8 (IAV/PR8). WT, or Tlr4−/− BMDCs were primed with PSA followed by infection with a GFP-expressing IAV/PR8 strain (PR8-GFP) at an MOI of 1, followed by flow cytometric analysis 24 h.p.i. (
To investigate the in vivo antiviral activity of PSA, WT or Ifnb1−/− ABX mice were administered PSA daily by oral gavage starting 4 days prior to and continuing to the day of infection with VSV. PSA treatment significantly reduced disease severity in WT ABX mice, with decreased daily and cumulative paralysis scores (
We have described a novel mechanism of immunomodulation by the commensal microbiota through IFNβ-mediated regulation of the homeostatic IFN-I response. Furthermore, we identified the mechanism by which a specific species of commensal bacteria regulates this response, signaling of B. fragilis glycolipids through TLR4 to induce expression of IFNβ by colon LP DCs. Importantly, this commensal-mediated regulation of IFNβ and the IFN-I response was found to play a critical role in maintaining health of the host by enhancing resistance to virus infection.
Our findings expand the analysis of ISG expression to multiple tissue sites as well as identifying which IFN-I family cytokine member is required for regulation of homeostatic ISG expression, a specific cellular source of commensal-induced IFNβ, and the molecular mechanism of IFNβ induction by a commensal microbial species. Indeed, ISG expression was reduced in Ifnb1−/− mice, with no additional reduction upon antibiotics treatment, specifically confirming the role of commensal-induced IFNβ in the homeostatic IFN-I response. Interestingly, among the IFN-I family members, IFNβ has been demonstrated to possess unique functionality, with increased anti-proliferative and regulatory function. IFN-I signaling can result in several different signaling outcomes all mediated through the same receptor. Importantly, the many different biological outcomes, whether pro- or anti-inflammatory, could be either beneficial or detrimental to the host depending on the context. The observed specific induction of IFNβ by the microbiota suggests controlled expression of the distinct IFN-Is to favor homeostatic expression only of the more regulatory variant, IFNβ.
Colon LP DCs were identified as a major source of IFNβ, with lower levels of IFNβ expression also detected in mLN DCs but not in splenic DCs. Intestinal DCs have previously been established to carry commensal antigen to the mLN, where they are restricted from entering into systemic immune circulation, and thus remain in the intestinal immune compartment. (Macpherson and Uhr, 2004) Our results are consistent with a model in which IFNβ is expressed in response to detection of microbial antigens by colon LP DCs, which then remain in the intestinal LP or traffic to the mLN. Despite this tight locational restriction of IFNβ expression, we observed systemic decreases in ISG expression in both Ifnb1−/− and antibiotics treated mice as well as functional effects of commensal induced-IFNβ during virus infection at an extra-intestinal site. It is possible that IFNβ secreted in the intestinal environment might enter the systemic circulation to mediate these effects. However, there were no detectable levels of IFNβ in the serum of WT SPF mice when analyzed by ELISA. IFNβ is extremely potent, capable of inducing ISG expression with a half-maximal effective concentration (EC50) of 0.2 pM. (Schreiber and Piehler, 2015) It is therefore possible that even a minute quantity of IFNβ, below the limits of detection of ELISA, might enter the circulation from the intestinal immune compartment and induce systemic effects on ISG expression. Alternatively, secretion of IFNβ by intestinal DCs might induce gene expression changes locally, in neighboring cells, which can then traffic to distal body sites. Further investigation will be required to determine which of these mechanisms accounts for systemic ISG expression under homeostatic conditions.
B. fragilis is a Gram-negative human commensal microbe, residing primarily in the lower GI tract. (Mazmanian et al., 2005) We discovered that the glycolipids of B. fragilis, PSA and LOS, signal through TLR4 to induce expression of IFNβ by DCs. Canonically, activation of TLR4 by bacterial LPS signals not only through TRIF to induce IFN-I signaling, but also through MyD88 to induce proinflammatory mediators including tumor necrosis factor α (TNFα), inducible nitric oxide synthase (iNOS), and IL-12. (Maeshima and Fernandez, 2013) Through activation of a robust and uncontrolled immune response, LPS exposure can cause severe systemic inflammation, leading to sepsis and in some cases potentially lethal septic shock and multi-organ failure. (Bohannon et al., 2013; Molinaro et al., 2015) As a result of its endotoxicity and the associated adverse effects, the clinical applications of LPS as an immunomodulator have been limited. However, most research on LPS has focused on E. coli. (Maeshima and Fernandez, 2013) Using numerous assays including proinflammatory cytokine secretion, the Limulus amoebocyte lysate (LAL) assay, rabbit pyrogenicity test, chick embryo lethality, and the Swartzman reaction, B. fragilis LOS has previously been demonstrated to have low endotoxic activity, suggesting it is a less inflammatory LPS species. (Erturk-Hasdemir et al., 2019; Kasper, 1976; Sveen et al., 1977) This difference in activity can be attributed to several structural differences between the two molecules. Whereas E. coli LPS is hexa-acylated, B. fragilis LOS represents a mixture of penta-, tetra-, and tri-acylated molecules. (Weintraub et al., 1989) In addition, the acyl-chains of B. fragilis LOS are longer, with C15-C17 fatty acid chains compared to C12-C14 fatty acids used by E. coli LPS. (Raetz et al., 2007; Weintraub et al., 1989) Finally, the diglucosamine backbone of B. fragilis LOS is mono- or non-phosphorylated compared to the bis-phosphorylated lipid A of E. coli. (Molinaro et al., 2015; Weintraub et al., 1989) Numerous studies have revealed that the number and length of the acyl-chains as well as the phosphorylation state of the disaccharide backbone in LPS are critical factors affecting the interaction with TLR4 and the downstream function of these molecules. (Maeshima and Fernandez, 2013) In this way, B. fragilis glycolipids might be able to induce the protective beneficial effects of IFNβ without the consequences of uncontrolled, pathologic inflammation.
Our findings suggest that induction of IFNβ through TLR4 signaling is not limited to B. fragilis but is a shared function of an entire class of commensal microbial molecules, Bacteroides LPS/LOS. Indeed, OM extracts from all of the Bacteroides sp. tested were able to induce IFNβ. OMs comprise numerous bacterial molecules, all of which have the potential to interact with immune cells and influence their response. (Kasper and Seiler, 1975; May and Grabowicz, 2018) Isolation of LPS/LOS from each of these bacterial species will therefore be required to further assess their IFNβ-inducing capabilities. Importantly, the genus Bacteroides makes up a large proportion of the human GI microbiome and is widespread in prevalence across the human population. (Salyers, 1984; Wexler, 2007) Indeed, Bacteroides LOS/LPS species might be the most abundant microbial molecules in the GI tract. As such, it is plausible that Bacteroides-induced IFNβ represents a ubiquitous and crucial mechanism by which commensal microbes communicate with their host to regulate the immune system and other physiological processes, ultimately contributing to human health.
The observed antiviral effect of commensal-induced IFNβ is mediated through priming of constitutive expression of ISGs, thus arming the immune system to respond immediately and robustly to virus infection. Due to the important role of IFN-I signaling and ISG expression in the context of the majority of mammalian virus infections, such a finding might represent a broad-spectrum, and therefore universally important, antiviral activity of the commensal microbiota. Current IFN-I based therapies rely on the administration of non-physiologic amounts of exogenous IFN-Is, which can have unwanted side effects. (Gottberg et al., 2000; Sleijfer et al., 2005) Delivery of an IFNβ-inducing microbial molecule thus represents a novel IFN-I-based therapeutic approach, which could enhance the IFN-I response while still being subjected to homeostatic regulatory mechanisms, reducing the potential for undesired side effects.
Conventional SPF mice of strains WT (C57BL/6, stock number 000664), Ifnar1−/− (B6.129S2-Ifnar1tmIAgt/Mmjax, stock number 010830), Tlr4−/− (B6.B10ScN-Tlr4Ips-deI/JthJ, stock number 007227), Myd88−/− (B6.129P2(SJL)-Myd88tm1,1Defr/J, stock number 009088), Trif−/− (C57BL/6J-Ticam1LPs2/L, stock number 005037) and IFNβ-YFP (B6.129-Ifnb1tm1Lky/J, stock number 010818) on a C57BL/6 background were purchased from Jackson Laboratory. The Ifnb1−/− (Ifnb1tm1(komp)vlcg) mouse strain used for this research project was created from ES cell clone 12782A-D4, generated by Regeneron Pharmaceuticals, Inc. and obtained from the KOMP Repository (www.komp.org). Mice were housed under SPF conditions. All genetically deficient mice and their respective controls were gender- and age-matched (typically 5-10 weeks). All experiments on animals were approved by the Harvard Medical Area Standing Committee on Animals (animal protocol number IS00000187-3). Germ free C57BL/6 mice were bred and maintained in sterile vinyl isolators in the animal facility at Harvard Medical School.
For studies involving depletion of the microbiota, SPF mice of the indicated strains were treated with a broad-spectrum antibiotics cocktail for 7 days. Vancomycin hydrochloride (0.5 g/L, Alfa Aesar), gentamicin sulfate (0.5 g/L, Sigma-Aldrich), and ampicillin (1 g/L, MP Biomedicals) were administered in the drinking water. Metronidazole (8 g/L, Alfa Aesar) and amphotericin B (0.1 g/L, Amresco Inc) were administered in 200 uL by OG once daily. Fecal samples were collected and plated under aerobic and anaerobic conditions to confirm microbiota depletion. For studies involving treatment with metronidazole alone, Metronidazole (0.5 g/L, Alfa Aesar) was administered in the drinking water for 7 days.
All bacteria used in this study were grown on Brucella blood agar plates or in liquid culture in rich Brain Heart Infusion Broth (BD) supplemented with Hemin (0.01%), vitamin K (0.1 mg/mL), glucose (0.5%), Monopotassium phosphate (0.5%), and L-cysteine (0.1%) in an anaerobic chamber (Coy Industries) at 37° Celsius (C). The following bacteria were used in this study B. fragilis (strain NCTC 9343, ATCC 25285), B. thetaiotaomicron (B. theta, ATCC 29741), B. vulgatus (ATCC 8482), B. dorei (DSM 17855), B. uniformis (ATCC 8492), B. ovatus (ATCC 8483), and C. ramosum (strain A031).
qRT-PCR Gene Expression Analysis
RNA was isolated from whole tissues or the DC+ and DC− fractions of spleens, mLNs, and colons following manufacturer's instructions with the RNeasy mini kit (Qiagen). cDNA was prepared with the Quantitect Reverse Transcription Kit (Qiagen). qRT-PCR was performed on a QuantStudio7 Flex or Pro Real-Time PCR system (Applied Biosystems) with RT2 SYBR Green ROX qPCR Mastermix (Qiagen). Amplification was achieved using an initial cycle of 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. Gene expression was normalized using Actb as a reference gene. The forward (f) and reverse (r) primers used to detect expression of the corresponding murine genes were as follows (SEQ ID NOs: 1-20, respectively):
Single-cell suspensions of the colonic lamina propria were prepared as previously described. (Dasgupta et al., 2014) Briefly, the tissue was cut transversely into approximately 2 cm pieces and then longitudinally to expose the lumen. To extract epithelial cells, tissue pieces were incubated in 1 mM dithiothreitol (Sigma) in PBS for 10 minutes, washed, and incubated in 30 mM Ethylenediaminetetraacetic acid (EDTA) three times for 8 minutes each. To digest the tissue, colon pieces were treated with RPMI medium supplemented with 5% fetal bovine serum (FBS) and 1 mg/mL collagenase type IV for 45 minutes at 37° C. in an atmosphere of 5% carbon dioxide (CO2) and then passed through a 70 μM filter. For single-cell suspensions of mLNs and spleens, tissues were harvested, treated with 1 mg/mL collagenase type IV in RPMI for 30 minutes at 37° C. in an atmosphere of 5% CO2, and passed through a 70 μm filter. Splenic samples were treated with red blood cell lysis buffer (Gibco).
Single-cell suspensions of the indicated tissues were prepared as described. DC positive and negative fractions were isolated with mouse pan-DC microbeads (Miltenyi), following the manufacturer's protocol.
GFWT C57BL/6 mice were administered B. fragilis strain NCTC 9343 or C. ramosum strain AO31 by oral gavage [200 uL, approximately 108-109 colony-forming units (CFU) per mouse]. Mice were housed in autoclaved cages and maintained on a diet of autoclaved food and water for 2 weeks and then euthanized for analysis.
For serum analysis, WT SPF or antibiotics treated mice were euthanized and blood was collected into 1.1 ml Z-Gel Micro Tubes (Sarstedt). Blood was left at room temperature for 30 minutes, centrifuged to remove coagulated cells [12,000 revolutions per minute (RPM), 10 minutes], and levels of IFNβ in the serum were quantified with the High Sensitivity Mouse IFN Beta ELISA kit (PBL Assay Science) according to the manufacturer's protocol. For cell culture supernatant analysis, cell free supernatants were collected from BMDC cultures and IFNβ levels were quantified with the Mouse IFN Beta ELISA kit (PBL Assay Science) according to the manufacturer's protocol.
DCs were derived from bone marrow of the indicated mice as previously described. (Helft et al., 2015) Briefly, bone marrow was collected from femurs, treated with red blood cell lysis buffer (Gibco), and cultured in 10 cm tissue culture (TC)-treated dishes at a density of 10×106 cells in 10 mL per plate in complete RPMI medium (cRPMI: RPMI 1640 supplemented with 10 mM HEPES, 10% fetal bovine serum, 2 mM L-glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin, and 50 μM 2-mercaptoethanol) with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). Cells were cultured for 6 days at 37° C. in an atmosphere of 5% CO2 with replenishment of medium on day 3. On day 6 the non-adherent and loosely adherent cells were harvested and plated at a density of 106 cells/mL in 200 uL per well of a 96-well round-bottom TC-treated cell culture plate for use in the indicated experimental procedures.
OM complexes were isolated from the indicated Bacteroides sp. as previously described. (Kasper and Seiler, 1975) Briefly, mid-log phase bacteria were pelleted, washed twice in 0.15 M sodium chloride (NaCl) and resuspended in a buffer of 0.15 M NaCl, 0.05 M sodium phosphate, and 0.01 M EDTA. The resuspended organisms were incubated for 30 minutes at 60° C. then subjected to mild shearing pressure by passing through a 25-gauge hypodermic needle. Cells were pelleted from the suspension and the supernatants were harvested and subjected to two rounds of centrifugation at 33,000 RPM for 2 hrs at 4° C. to pellet the OM extracts.
B. fragilis Glycolipid and Polysaccharide Purification
PSA was purified from B. fragilis mutant strain 444 and LOS was purified from an acapsular B. fragilis mutant strain by phenol-water extraction as previously described. (Tzianabos et al., 1992) To obtain the purified delipidated polysaccharide domain of PSA, PSA was subjected to acid hydrolysis with 2% acetic acid at 90° C. for 2 hours. The sample was neutralized the lipid was removed using chloroform/methanol extraction.
The following mouse-specific IgG monoclonal antibodies were purchased from Biolegend: Pacific Blue-conjugated anti-CD45, PE-conjugated anti-CX3CR1, PerCP-Cy5.5 conjugated anti-CD11b, PE-Cy7-conjugated anti-CD11c, and APC-conjugated anti-CD103. Fixable Viability Dye eFluor780 was purchased from eBioscience. Single-cell suspensions were stained with a suitable combination of fluorochrome-conjugated antibodies and fixable viability dye. Cells were fixed in 2% paraformaldehyde in PBS, and examined with an LSR II flow cytometer (BD). The data were analyzed with FlowJo software.
Mice were anesthetized by IP injection of ketamine and xylazine. 1×106 PFU of VSV strain Indiana was delivered in 10 uL per mouse by subcutaneous injection into the footpad as previously described. (Fensterl et al., 2014) Body weight and clinical disease score were monitored daily for 14 days following infection. VSV-infected mice exhibit an ascending paralytic disease that was scored as follows: 0=no symptoms, 1=tail paralysis, 2=hind-limb weakness, 2.5=paralysis of one hind limb, 3=paralysis of both hind limbs, 3.5=paralysis of both hind limbs and forelimb weakness, 4=forelimb and hind-limb paralysis, 5=moribund). Mice with a body condition score ≤2 or a disease score ≥4 were humanely euthanized.
On day 6 of culture, GM-CSF BMDCs were plated at a density of 106 cells/mL in 200 uL of cRPMI medium per well of a 96-well round-bottom TC-treated cell culture plate with 20 ng/mL GM-CSF and the indicated treatments for 24 hrs, washed, and resuspended in cRPMI medium containing either IAV/PR8-GFP or VSV-GFP at an MOI of 1. Cells were harvested for flow cytometric analysis 24 hrs post infection.
IFNβ neutralization
Mice were administered two doses of 250 ug IFNβ neutralizing antibody (anti-IFNβ, clone HDβ-4A7; Leinco Technologies) or Mouse IgG2a isotype control antibody (Leinco Technologies) by IP injection, 72 hrs and 24 hrs prior to infection with 106 PFU of VSV strain Indiana.
Information regarding statistical details and methods for each experiment can be found in the figure legends. Data is presented as mean +/− SEM unless otherwise indicated. Statistical analysis was performed using GraphPad Prism 7.0.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
This application claims the benefit of the filing dates of U.S. Provisional Application Nos. 63/039432 filed on Jun. 15, 2020, 63/039,843 filed on Jun. 16, 2020, and 63/085,392 filed on Sep. 30, 2020, the entire contents of which are incorporated by reference herein.
This invention was made with Government support under W81XWH1910625 awarded by the Department of Defense and AI120269 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/037310 | 6/15/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63085392 | Sep 2020 | US | |
| 63039843 | Jun 2020 | US | |
| 63039432 | Jun 2020 | US |
